Angiogenesis is one of the main targets of new anti-cancer drugs. We have discovered a novel anti-angiogenic substance, anastellin. The main characteristic of anastellin, which is a small fragment of fibronectin, is that it causes polymerization of fibronectin. The polymeric fibronectin generated in this manner (sFN) suppresses angiogenesis and blocks metastasis of various cancers in mice. Anastellin can be administered systemically either premixed with fibronectin or by injecting anastellin alone, which presumably polymerizes plasma fibronectin to form the active moiety. No toxicity has been observed in mice treated with systemic anastellin over long periods of time. At least two characteristics of anastellin/sFN seem to contribute to the anti-tumor effect: an anti-angiogenic activity and an ability to accelerate the elimination of circulating tumor cells. There are several other known anti-angiogenic proteins, and it is intriguing that they share some properties with anastellin. These proteins are heparin-binding fragments of extracellular matrix proteins (endostatin, thrombospondin, tumstatin) or of proteins involved in blood clotting (angiostatin, antithrombin). They all bind proteins that interact with the alphavbeta3 integrin (aVb3) or directly interact with this integrin, which is essential to the survival of angiogenic endothelial cells in the adult. In this application, we propose an experimental test of the hypothesis that anastellin and the other known protein inhibitors of angiogenesis share a common mechanism of action, and that this mechanism is to form protein complexes that bind to the aVb3 integrin and induce apoptosis. We will test this hypothesis by using angiogenesis models in knockout mice and by investigating the effect of fibronectin and vitronectin polymers on endothelial cells in vitro. We will also determine whether the angiogenesis inhibitors affect the incorporation of bone marrow-derived endothelial cell progenitors to colonize angiogenic sites. This study may provide important information on the mechanism of action of a class of angiogenesis inhibitors, information that may prove helpful in designing clinical trials for these compounds. [unreadable] [unreadable]